This invention relates to DNA sequences, recombinant DNA molecules and processes for producing interferon and interferon-like polypeptides. More particularly, the invention relates to DNA sequences expressed in appropriate host organisms. The recombinant DNA molecules disclosed herein are characterized by DNA sequences that code for polypeptides having an immunological or biological activity of human leukocyte interferon As will be appreciated from the disclosure to follow, the DNA sequences, recambinant DNA molecules and processes of this invention may be used in the production of polypeptides useful in antiviral and antitumor or anticancer agents and methods.
In this application the interferon nomenclature announced in Nature, 286, p. 2421 (Jul. 10, 1980) will be used. This nomenclature replaces that used in our earlier applications from which this application claims priority. E.g., IF is now designated IFN and leukocyte interferon is now designated IFN-xcex1;
Two classes of interferons (xe2x80x9cIFNxe2x80x9d) are known to exist. Interferons of Class I are small, acid stable (glyco)-proteins that render cells resistant to viral infection (A. Isaacs and J. Lindenmann, xe2x80x9cVirus Interference I. The Interferonxe2x80x9d, Proc. Royal Soc. Ser. B., 147, pp. 258-67 (1957) and W. E. Stewart, II, The Interferon System, Springer-Verlag (1979) (hereinafter xe2x80x9cThe Interferon Systemxe2x80x9d)). Although to some extent cell specific (The Interferon System, pp. 135-45), IFNs are not virus specific. Instead IFNs protect cells against a wide spectrum of viruses.
Human interferons (xe2x80x9cHuIFNxe2x80x9d) have been classified into three groups xcex1, xcex2 and xcex3. HuIFN-xcex1 or leukocyte interferon is produced in human leukocyte cells and together with minor amounts of HuIFN-p (fibroblast interferon) in lymphoblastoid cells. HuIFN-xcex2 has been purified to homogeneity and characterized (e.g. M. Rubenstein et al., xe2x80x9cHuman Leukocyte Interferon: Production, Purification To Homogeneity And Initial Characterizationxe2x80x9d Proc. Natl. Acad. Sci. USA, 76, pp. 640-44 (1979)). It is heterogeneous in regard to size presumably because of the carbohydrate moiety. Two components have been described, one of 21000 to 22000 and the other of 15000-18000 molecular weight. The component of lower molecular weight has been reported to represent a non-glycosylated form. The smaller form of HuIFN-xcex1 has also been reported to retain most or all of its HuIFN-xcex1 activity (W. E. Stewart, II et al., xe2x80x9cEffect Of Glycosylation Inhibitors On The Production And Properties of Human Leukocyte Interferonxe2x80x9d, Virology, 97, pp. 473-76 (1979)). A portion of the amino acid sequence of HuIFN-xcex1 from lymphoblastoid cells and its amino acid composition have been reported (K. C. Zoon et al., xe2x80x9cAmino Terminal sequence Of The Major Component Of Human Lymphoblastoid Interferonxe2x80x9d, Science, 207, pp. 527-28 (1980) and M. Hunkapiller and L. Hood, personal communication (1980))
HuIFN-xcex1 has also been reported to exist in several different forms, e.g. British patent application 2,037,296A. These forms appear to differ from each other structurally and physiologically. No accepted nomenclature has been adopted for these forms of HuIFN-xcex1. Therefore, in this application each form will be referred to by a number after the general HuIFN-xcex1 designation, i.e., HuIFN-xcex11 or HuIFN-xcex13.
HuIFN-xcex1 may, like many human proteins, also be polymoiphic. Therefore, cells of particular individuals may produce HuIFN-xcex1 species within the more general HuIFN-xcex1 group or forms within that group which are physiologically similar but structurally slightly different than the group or form of which it is a part. Therefore, while the protein structure of an HuIFN-xcex1 may be generally well-defined, particular individuals may produce a HuIFN-xcex1 that is a slight variation thereof, this allelic variation probably being less severe than the difference between the various forms of HuIFN-xcex1.
HuIFN is usually not detectable in normal or healthy cells (The Interferon System, pp. 55-57). Instead, the protein is produced as a result of the cell""s exposure to an IFN inducer. IFN inducers are usually viruses but may also be non-viral in character, such as natural or synthetic double-stranded RNA, intracellular microbes, microbial products and various chemical agents. Numerous attempts have been made to take advantage of these non-viral inducers to render human cells resistant to viral infection (S. Baron and F. Dianzani (eds.), Texas Reports On Biology And Medicine, 35 (xe2x80x9cTexas Reportsxe2x80x9d), pp. 528-40 (1977)). These attempts have not been very successful. Instead, use of exogenous HuIFN itself is now preferred.
Interferon therapy against viruses and tumors or cancers has been conducted at varying dosage regimes and under several modes of administration (The Interferon System, pp. 305-321). For example, interferon has been effectively administered orally, by innoculationxe2x80x94intravenous, intramuscular, intranasal, intradermal and subcutaneousxe2x80x94, and in the form of eye drops, ointments and sprays. It is usually administered one to three times daily in dosages of 104 to 107 units. The extent of the therapy depends on the patient and the condition being treated. For example, virus infections are usually treated by daily or twice daily doses over several days to two weeks and tumors and cancers are usually treated by daily or multiple daily doses over several months or years. The most effective therapy for a given patient must of course be determined by the attending physician who will consider such well known factors as the course of the disease, previous therapy, and the patient""s response to interferon in selecting a mode of administration and dosage regime.
As an antiviral agent, HuIFN has been used to. treat the following: respiratory infections (Texas Reports, pp. 486-96); herpes simplex keratitis (Texas Reports, pp. 497-500); acute hemorrhagic conjunctivitis (Texas Reports, pp. 501-10); varicella zoster (Texas Reports, pp. 511-15); cytomegalovirus infection (Texas Reports, pp. 523-27); and hepatitis B (Texas Reports, pp. 516-22). See also The Interferon System, pp. 307-19. However, large scale use of IFN as an antiviral agent requires larger amounts of IFN than heretofore have been available.
HuIFN has other effects in addition to its antiviral action. For example, it antagonizes the effect of colony stimulating factor, inhibits the growth of hemopoietic colony-forming cells and interferes with the normal differentiation of granulocyte and macrophage precursors (Texas Reoorts, pp. 343-49). It also inhibits erythroid differentiation in DMSO-treated Friend leukemia cells (Texas Reports, pp. 420-28). HuIFN may also play a role in regulation of the immune response. Depending upon the dose and time of application in relation to antigen, HuIFN-xcex1 can be both immunopotentiating and immunosuppressive in vivo and in vitro (Texas Reports, pp. 357-69). In addition, specifically sensitized lymphocytes have been observed to produce HuIFN-xcex1 after contact with antigen. Such antigen-induced HuIFN-xcex1 could therefore be a regulator of the immune response, affecting both circulating antigen levels and the expression of cellular immunity (Texas Reports, pp. 370-74). HuIFN is also known to enhance the activity of killer lymphocytes and antibody-dependent cell-mediated cytotoxicity (R. R. Herberman et al., xe2x80x9cAugmentation By Interferon Of Human Natural And Antibody Dependent Cell-Mediated Cytotoxicityxe2x80x9d, Nature, 277, pp. 221-23 (1979); P. Beverley and D. Knight, xe2x80x9cKilling Comes Naturallyxe2x80x9d, Nature, 278, pp. 119-20 (1979); Texas Reports, pp. 375-80). Both of these species are probably involved in the immunological attack on tumor cells.
Therefore, in addition to its use as a human antiviral agent, HuIFN has potential application in antitumor and anticancer therapy (The Interferon System, pp. 319-21). It is now known that IFNs affect the growth of many classes of tumors in many animals (The Interferon System, pp. 292-304). They, like other antitumor agents, seem most effective when directed against small tumors. The antitumor effects of animal IFN are dependent on dosage and time but have been demonstrated at concentrations below toxic levels. Accordingly, numerous investigations and clinical trials have been and continue to be conducted into the antitumor and anticancer properties of IFNs. These include treatment of several malignant diseases such as osteosarcoma, acute myeloid leukemia, multiple myeloma and Hodgkin""s disease (Texas Reports, pp. 429-35). In addition, HuIFN has recently been shown to cause local tumor regression when injected into subcutaneous tumoral nodules in melanoma- and breast carcinoma-affected patients (T. Nemoto et al., xe2x80x9cHuman Interferons And Intralesional Therapy of Melanoma And Breast Carcinomaxe2x80x9d, Amer. Assoc. For Cancer Research, Abs. nr. 994, p. 246 (1979)). Although the results of these clinical tests are encouraging, the antitumor and anticancer applications of IFN have been severely hampered by lack of an adequate supply of purified IFN.
Today, HuIFN-xcex1 is produced either through human cells grown in tissue culture or through human leukocytes collected from blood donors. 2.6xc3x97109 IU of crude HuIFN-xcex1 have been reported from 800 1 of cultured Namalva cells (P. J. Bridgen et al., supra). At very large blood centers, eg., the Finnish Red Cross Center in Helsinki, Finland, the production capacity is about 1011 IU of crude HuIFN-xcex1 annually. Since dosage is typically 3xc3x97106 IU per patient per day, these sources are not adequate to provide the needed commercial quantities of HuIFN-xcex1. Therefore, production of HuIFN-xcex1 by other procedures is desirable. Because the specific activity of IFN-xcex1 is high, in the order of 4.0xc3x97108 to 109 IU/mg, the amount of HuIFN-xcex1 required for commercial applications is low. For example, 100 grams of pure HuIFN-xcex1 would provide between 3 and 30 million doses.
Recent advances in molecular biology have made it possible to introduce the-DNA coding for specific nonbacterial eukaryotic proteins into bacterial cells. In general, with DNA other than that prepared via chemical synthesis, the construction of such recombinant DNA molecules comprises the steps of producing a single-stranded DNA copy (cDNA) of a purified messenger RNA (IRNA) template for the desired protein; converting the cDNA to double-stranded DNA; linking the DNA to an appropriate site in an appropriate cloning vehicle to form a recombinant DNA molecule and transforming an appropriate host with that recombinant DNA molecule. Such transformation may permit the host to produce the desired protein.
Several non-bacterial proteins and genes have been obtained in E. coli using recombinant DNA technology. These include a protein displaying rat proinsulin antigenic determinants (L. Villa-Komaroff et al., xe2x80x9cA Bacterial Clone Synthesizing Proinsulinxe2x80x9d, Proc. Natl. Acad. Sci. USA, 75, pp. 3727-31 (1978)), rat growth hormone (P. H. Seeburg et al., xe2x80x9cSynthesis Of Growth Hormone By Bacterialxe2x80x9d, Nature, 276, pp. 795-98 (1978)), mouse dihydrofolate reductase (A. C. Y. Chang et al., xe2x80x9cPhenotypic Expression In E. coli Of A DNA Sequence Coding For Mouse Dihydrofolate Reductasexe2x80x9d, Nature, 275, pp. 617-24 (1978)), human somatostatin (K. Itakura et al., xe2x80x9cExpression In Escherichia coli Of A Chemically Synthesized Gene For The Hormone Somatostatinxe2x80x9d, Science, 198, pp. 1056-63 (1977)); European patent applications 0,001,929, 0,001,930, and 0,001,931 and cognate applications in other countries), the A and B polypeptide chains of-human insulin (D. V. Goeddel et al., xe2x80x9cExpression In Escherichia coli Of Chemically Synthesized Genes For Human Insulinxe2x80x9d, Proc. Natl. Acad. Sci. USA, 76, pp., 106-10 (1979) and the European and related patent specifications, supra), antigens of human hepatitis B virus (C. J. Burrell et al., xe2x80x9cExpression In Escherichia coli: Of Hepatitis B Virus DNA Sequences Cloned In Plasmid pBR322xe2x80x9d, Nature, 279, pp. 43-7 (1979) and M. iasek et al., xe2x80x9cHepatitis B Virus Genes And Their Expression In E. colixe2x80x9d, Nature, 282, pp. 575-79 (1979).), human growth hormone (D. V. Goeddel et al., xe2x80x9cDirect Expression In Escherichia coli Of A DNA Sequence Coding For Human Growth Hormonexe2x80x9d, Nature, 281, pp. 544-51 (1979)), SV40 t antigen (T. M. Roberts et al., xe2x80x9cSynthesis of Simian virus 40 t Antigen In Escherichia colixe2x80x9d, Proc. Natl. Acad. Sci. USA, 76, pp. 5596-600 (1979)), and human fibroblast interferon (HuIFN-xcex2) (T. Taniguchi et al., xe2x80x9cConstruction And Identification Of A Bacterial Plasmid Containing The Human Fibroblast Interferon Gene Sequencexe2x80x9d, Proc. Japan Acad., 55, Ser. B, pp. 464-69 (1979) together with personal communication 1980).
None of these recombinant DNA processes, however is directed, as is this invention, toward the synthesis of HuIFN-xcex1. This is the problem to which the present invention is addressed. Its solution is not facilitated as were the above described recombinant DNA schemes by the availability of the sequence information required to prepare a synthetic gene (e.g., somatostatin) or of a cell type or virus rich in aparticular DNA sequence (e.g., hepititis viral antigen) or URNA species (e.g., rat insulin) which allows preparation and identification of bacterial clones containing the desired hybrid DNA, or of a system allowing the selection of E. coli hosts that express the desired protein (e.g., mouse dihydrofolate reductase). Neither is it aided by the report of a plasmid which is said to contain a DNA sequence that hybridizes to a mRNA from a poly(A) RNA, that mRNA producing HuIFN-xcex2 activity in oocytes (e.g., fibroblast interferon). Nor is the solution of the present invention addressed as is the apparent suggestion of Research Disclosure No. 18309, pp. 361-62 (1979) to preparing pure or substantially pure HuIFN-xcex1mRNA before cloning of the HuIFN-xcex1 gene.
Finally, it should be recognized that the selection of a DNA sequence or the construction of a recombinant DNA molecule which hybridizes to d mRNA from. polyA RNA, that mRNA producing HuIFN activity in oocytes, is not sufficient to demonstrate that the DNA sequence or the hybrid insert of the recombinant DNA molecule corresponds to HuIFN. Instead, only the production of a polypeptide that displays an immunological or biological. activity of HuIFN can actually demonstrate that the selected DNA sequence or constructed recombinant DNA molecule corresponds to HuIFN. More importantly, it is only after HuIFN activity is shown that the DNA sequence, recombinant DNA molecule or sequences related to them may be employed to select other sequences corresponding to HuIFN in accordance with this invention.
It will therefore be appreciated from the foregoing that the problem of producing HuIFN-xcex1 with the use of recombinant DNA technology is much different than any of the above described processes. Here, a particular DNA sequence of unknown structurexe2x80x94that coding for the expression of HuIFN-xcex1 in an appropriate hostxe2x80x94must be found in and separated from a highly complex mixture of DNA sequences in order for it to be used in the production of HuIFN-xcex1. Moreover, this location and separation problem is exacerbated by the predicted exceedingly low concentration of the desired DNA sequence in the complex mixture and the lack of an effective means for rapidly analyzing the many DNA sequences of the mixture to select and separate the desired sequence.
The present invention solves the problems referred to by locating and separating DNA sequences that code for the expression of HuIFN-xcex1 in an appropriate host and thereby providing DNA sequences, recombinant DNA molecules and methods by means of which a host is transformed to produce a polypeptide displaying an immunological or biological activity of human leukocyte interferon.
By virtue of this invention, it is possible to obtain polypeptide(s) displaying an immunological or biological activity of HuIFN-xcex1 for use in antiviral, antitumor or anticancer agents and methods. This invention allows the production of these polypeptides in amounts and by methods hitherto not available.
As will be appreciated from the disclosure to follow, the DNA sequences and recombinant DNA molecules of the invention are capable of directing the production, in an appropriate host, of a polypeptide displaying an immunological or biological activity of HIFN-xcex1. Replication of these DNA sequences and recombinant DNA molecules in an appropriate host also permits the production in large quantities of genes coding for these polypeptides. The molecular structure and properties of these polypeptides and genes may be readily determined. The polypeptides and genes are useful, either as produced in the host or after appropriate derivatization or modification, in compositions and methods for detecting and improving the production of these products themselves and for use in antiviral and antitumor or anticancer agents and methods.
This process is therefore distinguishable from the prior processes, above mentioned, in that this process, contrary to the noted prior processes, involves the preparation and selection of DNA sequences and recombinant DNA molecules which contain appropriate DNA sequences which code for at least one polypeptide displaying an immunological or biological activity of HuIFN-xcex1.
It will be appreciated from the foregoing that a basic aspect of this invention is the provision of a DNA sequence which is characterized in that it codes for a polypeptide displaying an imunological or biological activity of HuIFN and is selected from the group consisting of the DNA inserts of Z-pBR322 (Pst)/HcIF-4c, Z-pBR322(Pst)/HcIF-2h, Z-pBR322(Pst)/HcIF-SN35, Z-pBR322(Pst)/HcIF-SN42, Z-pKT287(Pst)/HcIF-2h-AH6, DNA sequences which hybridize to any of the foregoing DNA inserts, DNA sequences, from whatever source obtained, including natural, synthetic or semi-synthetic sources, related by mutation, including single or multiple, base substitutions, deletions, insertions and inversions to any of the foregoing DNA sequences or inserts, and DNA sequences comprising sequences of codons which on expression code for a polypeptide displaying similar immunological or biological activity to a polypeptide coded for on expression of the codons of any of the foregoing DNA sequences and inserts and that these sequences permit the production of interferon and interferon-like polypeptides in hosts.